Typically, clinical immunoassay procedures involve the mixing of a dilute sample of blood serum containing a trace of an antigenic species of interest with an aliquot of antibody prepared to react specifically and exclusively with the trace antigen. The antibody is usually added in moderate stoichiometric excess over the maximum amount of antigen expected to be present in the most abnormal of natural occurring samples.
The reaction between these two species of molecules is bimolecular and its rate is proportional to the product of their concentrations. When the concentration of an antigen is as low as 10.sup.-12 gm/ml (as is common for many naturally occurring "hormones"), the time for completion of the reaction can be longer than 24 hours. This leads to inconveniently long assay procedures.
In addition, at such low concentrations, the ability to detect evidence of the reaction is severely compromised.
Variations of the famous isotope dilution technique are used for such assays. In its simplest form, a sample of radioactive isotopically labelled antigen is added to the mixture in an amount in slight excess of a stoichiometric equivalent of the added antibody. The labelled antigen competes with the unlabelled antigen for antibody and is bound in proportion to the ratio of labelled to unlabelled antigens. After the reaction is complete, the reacted antigen-antibody complex is separated (by any one of a large number of methods) from the unreacted antigen, and the radioactivity of either or both separated fractions can be used to determine the initial concentration of antigen in the serum sample.
It is clear that other labelled antigens (e.g., fluorescent-labelled, chemiluminescent-labelled, spin-labelled, etc.) can be used in similar kinds of assays. In general, the ability of alternative methods to compete in sensitivity with the radio-labelled method depends upon the sensitivity of the assay procedure for fluorescence, chemiluminescence, etc. In contrast to radioactivity, the sensitivity of many of the methods for detecting these other labels are themselves highly concentration dependent.
Where non-isotopic methods are comparable in sensitivity to, or are greater in sensitivity than, the radio-isotopic dilution methods, they are often preferred. The health hazard associated with exposure of laboratory personnel to radioactive substances is absent with the use of non-radioactive labels. In addition, longer reagent life is available with non-isotopic labels because radioactive immunoassay reagents typically have finite and short half-lives. Also, the lower cost of non-radioactive reagents and the simpler and less costly instruments for detecting them, makes non-radioactive labels more desirable.
Therefore, an immunoassay method using non-radioactive labeling which automatically concentrates both the antigen and antibody 300 or more times (and therefore increases the rate of reaction 90,000 or more times) would provide an enormous advantage over existing methods which are highly time consuming, e.g., Radioimmunoassay & Related Techniques Methodology & Clinical Application by Thorell & Larson, published by C. V. Mosby Co., St. Louis, 1978, pp. 144, 186, 198, 200.
Despite the fact that preconcentrating has been recognized as desirable, to date there is no convenient means of accomplishing this result. Reactants can be preconcentrated by centrifugation, but this technique is not entirely satisfactory. In the first instance, the preconcentrated reactants often must be partly rediluted when they are removed from the centrifuge for reaction purposes. Secondly, this procedure usually requires expensive equipment. Thirdly, the procedure is inordinately time consuming. This is especially so for species of low molecular weight such as small antigens like angiotensin and haptens.
The invention achieves preconcentration of both reactants of the reaction in the very medium in which the reaction is accomplished. This eliminates the aforementioned drawback of redilution. In addition, the invention causes the reactants to preconcentrate at concentrations many times greater than generally achievable in prior art methods as applied to immunoassays. Furthermore, the invention seeks to achieve all these objectives at low cost, and in a rapid manner.
An assaying technique of the prior art features the reaction of immunoreactants, antigen (or hapten) on one hand, and antibody on the other, within a localized zone of a gel medium. The test substance is caused to migrate by electrophoresis through the gel into reactive contact with the immobilized reactant. After equilibration takes place, the unreacted or unbound substances are separated by further electrophoresis away from the immobilized reactant. Such a system as described above, may be seen with reference to D. W. Renn et al U.S. Pat. No. 3,966,897 issued June 29, 1976. This invention, however, does not teach how the reactants may be preconcentrated within the gel reaction medium.
The present invention is generally distinguished from the prior immunoassay art by means of concentrating the constituents of a reaction in the same medium in which they react. In the prior art, the ultimate potential for achieving extremely rapid reaction rates is never fully realized. This is because the reactants are not brought together in concentrated form.